Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/805—Optical property
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/806—Electrical property or magnetic property

Definitions

• the invention relates to a device comprising a biologically active substance immobilized on a covalent nitride substrate by a bifunctional binding agent.
• the ability to dose a substance or mixture of substances in a given medium is of great interest in various applications such as process monitoring, quality control, determination of polluting substances in air and water, determination of analytes in physiological fluids, diagnosis of diseases, histological compatibility between individuals, quality of food, etc.
• concentrations of substances to be measured range from molar to picomolar quantities.
• nitrides such as boron nitride (BN), titanium nitrides, phosphorus nitrides such as P 3 N 5 , carbon nitrides, silicon nitrides and metal nitrides of transition.
• silicon nitride because of its refractive index, its waveguide properties, its properties of low optical absorption, and its chemical inertness, which make it well suited to applications using integrated optics.
• the invention therefore aims to provide devices comprising a biologically active substance immobilized on a covalent nitride substrate.
• the invention relates to a device comprising a substrate and at least one biologically active substance linked to at least part of the surface of this substrate, obtained by reaction, simultaneous or sequential, of said substrate and of said substance with an agent for bifunctional bond, one of the functions of which is photoactivatable and generates carbenes and serves to bind the binding agent to the mineral substrate, and the other function serves to bind the binding agent to the biologically active substance, characterized in that said substrate is a covalent mineral nitride.
• the bifunctional linker must have one or more functional groups capable of generating carbene radicals by exposure to light (visible or UV). Examples of such functions are diazirine functions.
• the other function of the liaison officer must be capable of reacting with the biologically active substance, in order to bind the binding agent to this substance.
• a preferred class of linkers is that of homo- or hetero-bifunctional agents.
• the homobifunctional photoactivatable binding agents carry at each end of the molecule a photosensitive functional group, preferably aryldiazirine functions for the functionalization of covalent nitrides.
• the distance between the photoactivatable aryldiazirine functions can, for example, range from 0.4 to 1.7 nm.
• the photoactivatable heterobifunctional linkers carry different functional groups at each end of the molecule and can be of various molecular sizes.
• One is selectively reactive with the biologically active substance while the other is a photoactivatable group, preferably an aryldiazirine or alkyldiazirine group.
• the group reactive with the biologically active substance can be in particular an isothiocyanate group, capable of reacting with primary amines, or an ester function of succinimidyloxy, capable of reacting with primary amines, or a maleimido group, the double bond of which can react with an addition reaction with thiols.
• Activated esters and anhydrides readily react with a hydroxy or amino group in an organic solvent or an organic solvent-buffer mixture, if necessary in the presence of a condensation catalyst.
• biologically active substance (SBA for short) must be understood in a broad sense and includes in particular synthetic or natural substances of low molecular weight such as phospholipids, glycolipids, oligo- and mono-saccharides, amino acids, peptides, oligonucleotides, drugs or metabolites. This can also be natural or synthetic high molecular weight macromolecules such as proteins, enzymes, antigens, haptens, antibodies, receptors, cell binding proteins, polycarbohydrates, or complex biological structures such as viruses , organelles, cells, bacteria or sections of tissue. As indicated above, these substances can be modified by the bonding agent before coating the surface or be immobilized on the surface after the photoimmobilisation of said agent on the surface.
• the SBA can be immobilized in the form of a substantially monomolecular layer by suitably regulating the number of molecules of the binding agent linked to the substrate by the photochemical process.
• This mixture can be obtained by varying the concentration of the bonding agent applied to the substrate and the conditions of exposure to radiation (duration, intensity).
• the exposure to activating radiation of the carbene generating function can be carried out globally on a surface of the material, or in a pattern of desired shape using an appropriate mask.
• the light source must emit radiation, the wavelength or wavelengths of which overlap with the absorption band of the photoactivatable function of the binding agent.
• a light source emitting at 350 ⁇ 30 nm has been found to be satisfactory.
• radiation with a wavelength greater than 320 nm it is advantageous to use radiation with a wavelength greater than 320 nm to avoid a possible harmful effect on the SBA.
• suitable sources are suitably filtered sunlight, polarized light, light from a commercially available source such as the Stratalinker® device, high pressure mercury lamps, lasers, etc.
• Patterned exposure can be achieved using an appropriate mask or by "writing" with a laser.
• the device of the invention can be, by way of non-limiting example, a biosensor, a bioreactor, an implant, a medical or industrial analysis device, or a diagnostic device.
• the coating can be carried out by application and drying, by physical adsorption or by sedimentation assisted by centrifugation, etc.
• Si chips coated with silicon nitride were rinsed and sonicated in acetone three times for 15 minutes and once in isopropanol for 15 minutes to remove the protective photosensitive coating. Then, the chips were dried under vacuum for 60 minutes (5 mbar, at room temperature). MAD (10 ⁇ l, 1 Mm in ethanol) was applied to the surface of the silicon nitride. The chips were dried in vacuo for 30 minutes (5 mbar, at room temperature). The chips were exposed to an activating light (Stratalinker® light source, 350 nanometers, 20 minutes, 0.7 mWcm -2 ) through masks with slit pattern (300 ⁇ m and 20 ⁇ m).
• an activating light Stratalinker® light source, 350 nanometers, 20 minutes, 0.7 mWcm -2
• T-BSA (10 ⁇ l of a solution containing 610 ⁇ g of protein per ml in 1.5 Mm NaCl, 0.5 Mm Na phosphate buffer, pH 7.4) was deposited on the surface of silicon nitride and dried under reduced pressure (2 hours, 5 mbar). The irradiation was carried out through a mask with a pattern of slits of 20 ⁇ m with the Stratalinker light source for 20 minutes and at an exposure dose of 0.7 Mwcm -2 .
• the chips were washed and treated with ultrasound three times for 15 minutes in 1.5 Mm NaCl, 0.05 Mm sodium phosphate, pH 7.4 buffer containing 0.02% Tween®.
• AFM atomic force microscopy
• the 20 ⁇ m patterned chips were mounted in the liquid cell of the atomic force microscope. After adding PBS, the system was allowed to equilibrate for 60 minutes before imaging by scanning atomic force microscopy. Here again we obtain a pattern corresponding to that of the slit mask.
• mouse immunoglobulin radiolabelled with [ 14 C].
• Mouse IgG (1 mg in 500 ⁇ l of 100 Mm sodium phosphate buffer, pH 6.8) was transferred to 0.1 M HEPES buffer, pH 7.5 by chromatography on a PD10 chromatographic column. Then 29.7 ⁇ l (1 ⁇ mol, 53 ⁇ Ci) of [ 14 C] -formaldehyde and 65 ⁇ mol of sodium cyanoborohydride were added to the protein collected (890 ⁇ g in 1.5 ml of HEPES buffer) and the mixture was stirred. reaction mixture for 4 hours at room temperature in the dark.
• the antibodies were mixed with T-BSA (BSA for the control samples) in a weight ratio of 1/4.
• Samples containing 0.25 ⁇ g of antibody and 1.0 ⁇ g of T-BSA were adjusted to a final volume of 25 ⁇ l (in 0.01 M sodium phosphate buffer, pH 6.8) and applied to 5 x 5 mm chip area.
• the chips were placed in the wells of a Falcon multiwell plate. After drying under reduced pressure (2 mbar) at room temperature the coated chips were irradiated for 20 minutes.
• a Stratalinker 2400 ultraviolet crosslinking device (Stratagene GmbH, Heidelberg) equipped with 5 bulbs (F158 BL, 15W black light) was used for activation of the photolabel.
• the samples were placed 4 cm away from the light source and exposed to a radiation dose of 0.7 Mwcm -2 (Suss model 1000 intensitometer with a detection cell at 320 nanometers).
• the silicon nitride chips were shaken perpendicular to the bulbs with a deflection of 4 cm and a frequency of 26 cycles per minute.
• Identically treated and non-irradiated samples served as a control.
• the modified chips were washed with PBS containing 0.02% Tween 20 (3 x 500 ⁇ l / chip), double distilled water (3 x 500 ⁇ l / chip).
• the assay for photoimmobilized antibodies was carried out with [ 14 C] radiolabelled antibodies. It was thus determined that approximately 20% of the antibodies applied were photoimmobilized on the silicon nitride surface.
• glucose oxidase ⁇ -D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4.
• GOD 81 mg
• ⁇ -D-glucose 1.27 g
• TRIMID 170 ⁇ l, 29 ⁇ mol in chloroform
• T-GOD (25 ⁇ l containing 2 ⁇ g of T-GOD in a 1.5 mM NaC1 buffer, 0.05 mM sodium phosphate, pH 7.4) was deposited on chips coated with polished silicon nitride and dried under reduced pressure at room temperature. Samples were irradiated by the Stratalinker light source for 40 minutes. Then the samples exposed to light and non-irradiated samples were washed by washing with PBS containing 0.02% Tween 20 (3 x 500 ⁇ l / chip) and double distilled water (3 x 500 ⁇ l / chip) . The activity of the glucose oxidase retained on the chips was determined by the spectrophotometric method described by Foulds et al.
• the assay is based on a peroxidase indicator reaction which measures the hydrogen peroxide released in the enzyme reaction.
• This particular dosage is based on the coupling oxidant of hydrogen peroxide with 4-aminophenazone and a phenol, in the presence of horseradish peroxidase to form a chromogen (a quinoneimine type dye) with a maximum absorption at 520 nanometers.
• the enzymatic activities measured were as follows:
• Cantilevers for scanning force microscopy were cleaned by ultrasonic treatment in acetone three times for 15 minutes and in isopropanol once for 15 minutes. 5 ⁇ l of MAD (100 ⁇ M in ethanol) was deposited three times on the cantilever tip, allowing the tip to air dry between applications. The samples were placed in the Stratalinker apparatus at a distance of 4 cm from the light source and exposed for 20 minutes to an irradiation dose of 0.7 mWcm -2 . As a variant, the cantilever tip was activated for 60 seconds with a focused argon laser (354 nm) (beam diameter 50 ⁇ m) with an irradiation dose of 130 mXcm -2 .
• the photoactivated cantilever tips were rinsed and treated with ultrasound three times for 1 minute in ethanol.
• the cantilever tips were then soaked in a solution containing freshly prepared fragments F (ab ') (100 ⁇ l, 51 ⁇ g of protein) in 0.1 M sodium acetate, 0.5 M sodium chloride, and 1 mM EDTA, pH 5.0 and incubated for 12 hours at 37 ° C.
• the fluorescein-labeled F (ab ') 2 fragments were treated with dithiothreitol.
• the reducing agent was eliminated by chromatography on a PD10 column and the F (ab ') fragments were thermochemically immobilized on tips functionalized with MAD. Then the cantilever tips were rinsed and treated with ultrasound in 0.1 M sodium phosphate buffer, pH 6.0 and allowed to equilibrate in this buffer for 2 hours. The binding of antibody fragments to the tip by MAD was visualized by fluorescence microscopy.

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• 1995
  • 1995-07-19 FR FR9508737A patent/FR2737012B1/fr not_active Expired - Fee Related
• 1996
  • 1996-07-18 DE DE69615766T patent/DE69615766T2/de not_active Expired - Lifetime
  • 1996-07-18 EP EP96401605A patent/EP0754947B1/de not_active Expired - Lifetime
  • 1996-07-19 US US08/684,458 patent/US5858802A/en not_active Expired - Lifetime

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